Augmentation of titer for vaccination in animals

ABSTRACT

The disclosure relates to a composition added to animal feed used in combination with a vaccine to enhance the effectiveness of the vaccine. Amongst other effects, the composition raises the titer of antibodies to the vaccine.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 13/400,520, filed Feb.20, 2012, which is a divisional of U.S. patent application Ser. No.11/380,359, filed Apr. 26, 2006, now U.S. Pat. No. 8,142,798, each ofwhich is incorporated herein by reference in its entirety.

FIELD

The invention relates to combinations of a composition and a vaccinethat augments titer of antibodies to the vaccine and methods for usingthe combination.

BACKGROUND

The immune response involves two distinct systems: the innate system andthe acquired (antibody-mediated) system. The innate system is anevolutionarily ancient system that uses a variety of strategies toprevent infection. These include the epithelial cell barriers providedby skin, the gastrointestinal tract and the linings of the lung andmammary gland. In addition, the innate system includes the acidicbarrier of the stomach (or abomasum in ruminant animals) and thedigestive enzymes of the stomach, pancreas and small intestine. Finally,the innate system includes the white blood cells, macrophages andneutrophils. These cells first recognize pathogens via the presentationof unique markers on the surface of pathogens and then phagocytose andkill pathogens.

The innate system provides the initial immune response and provides thetime required for the acquired antibody system to respond and to developthe antibodies needed to combat a specific pathogen. Usually one week toseveral weeks are required for a person or an animal to develop anantibody response. In this intervening time, an organism depends uponthe innate system to hold off infection.

The acquired immune system may develop antibodies in response to aspecific pathogen, toxin, chemical or any molecule that the organismrecognizes as an antigen (i.e. the immune system recognizes the antigenas non-self). When pathogens infect a person or an animal, specificcellular markers associated with the pathogen are presented toantibody-producing cells. The acquired immune system then undergoes aprocess termed “clonal expansion”. Specifically, this allows for themass production of cells which produce antibodies which are directedtoward a specific antigen associated with the pathogen.

Antibodies are synthesized by T-cells and B-cells. The T-cells mature inthe thymus and present antibodies that are bound to their extracellularsurfaces. The T-cells then circulate freely in blood and throughlymphatic tissues. The binding of the T-cell to a pathogen via the boundantibody thereby results in the identification and subsequentdestruction of the pathogen. In contrast, the antibodies produced byB-cells are secreted into the blood where they circulate freely. WhenB-cell-produced antibodies bind to a pathogen, they initiate a cascadeof events which results in the identification and killing of thepathogen. Antibodies which are produced in response to immunization areclassed into antibody isotypes. The three most important antibodyisotypes include IgM, IgG1 and IgG2. Other isotypes include, but are notlimited to the IgA, IgD, IgG3, IgG4 and IgE isotypes and, withinpoultry, the IgY isotype.

The adaptive IgM response is the first antibody produced by T- andB-cells in response to an antigen; however, it is a relatively “weak”antibody with limited affinity for antigen and specificity. More“powerful” antibody responses are contained within the IgG1 and IgG2isotypes; however, the development of the IgG1 and IgG2 isotypesrequires longer periods of time. IgG isotype responses in pregnantindividuals are particularly important as these are the antibodyisotypes which are transferred from mother to offspring via colostrum attime of birth and which thereby transfer passive immunity to thenewborn.

Vaccination (also called immunization) against disease is commonlypracticed within the human medical and livestock industries. Forexample, to vaccinate against a pathogen, an animal is administered avaccine in the form of non-infectious version of the pathogen or isadministered only a portion of the pathogen. The acquired immune systemresponds by producing antibodies to the vaccine. If the animal issubsequently exposed to the live pathogen, the antibodies made inresponse to the vaccine are quickly mobilized and then recognize andtarget the pathogen for destruction.

The livestock industry relies upon immunization protocols againstlivestock-specific diseases to minimize morbidity and mortality arisingfrom fungal, viral and bacterial infections. For example, in the dairyindustry, it is common to vaccinate animals against E. coli as this isone of the most common forms of mammary gland infections (i.e. mastitis)and causes loss of production and, in severe cases, loss of the cow.

Several problems arise from current vaccination protocols. For example,the efficacy of vaccination protocols varies from individual toindividual. Specifically, some individuals will develop a high titer(high serum concentration of antibodies) in response to a specificimmunization protocol whereas others do not. As there is a directcorrelation between the titer of an antibody and the immune system'sresponse to an infection, some individuals remain susceptible to aninfection by the pathogen even though they have been vaccinated.Consequently, there remains a need to improve the effectiveness ofvaccines to reduce incidence of disease in animal populations.

SUMMARY

The disclosure relates to combinations for enhancing the effectivenessof vaccines and methods for using the combinations. The combinations ofthe disclosure use a composition that has the following constituents:β-1,3 (4)-endoglucanohydrolase, β-1,3 (4)glucan, silica, mineral clayand mannans. This composition is fed to animals that are about toundergo a vaccination protocol or are undergoing a vaccination protocol.

The combinations increase the effectiveness of the vaccine by increasingthe serum concentration (titer) of antibodies to the vaccine. Theincreased serum concentration of antibodies remains even after thecomposition is withdrawn from the diet of the animals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results from Western blotting experiments that demonstratethe effect of the composition on the expression of neutrophil L-selectinas described in Example 1.

FIG. 2 shows results from Western blotting experiments that demonstratethe effects of the composition in unheated and heated (pelleted) formson the expression of neutrophil L-selectin as described in Example 2.

FIG. 3 is a graph summarizing the effects of the composition on theexpression of the mRNA encoding L-selectin in rat neutrophils asdescribed in Example 3.

FIG. 4 shows results from Western blotting experiments that demonstratethe effects of the composition on the expression of neutrophilinterleukin-1β (Il-1β) as described in Example 4.

FIG. 5 is a bar graph that summarizes the results of ELISAs used toquantitate IgG1 titer against the J5 vaccine as described in Example 6.

FIG. 6 is a bar graph that summarizes the results of ELISAs used toquantitate IgG2 titer against the J5 vaccine as described in Example 6.

FIG. 7 is a bar graph that illustrates the effects of a low dose (15grams/day) and high dose (30 grams/day) of the composition on theexpression of interleukin-113 in beef cattle as described in Example 6.

FIG. 8 shows results from Western blotting experiments that demonstrateeffects of the composition on expression of neutrophil L-selectin inbeef cattle as described in Example 6.

FIG. 9 is a bar graph that summarizes the results of ELISAs used toquantitate the total of the IgG1 and IgG2 titer as described in Example7.

DETAILED DESCRIPTION

As required, detailed embodiments of the present composition aredisclosed herein; however, it is to be understood that the disclosedembodiments are merely exemplary of the invention, which may be embodiedin various forms. Therefore, specific details disclosed herein are notto be interpreted as limiting, but merely as a basis for the claims andas a representative basis for teaching one skilled in the art tovariously employ the present composition in virtually any appropriatemanner.

The present disclosure is addressed to combinations that augment immunefunction in animals and methods for using the combinations. Generally,the combinations of the disclosure use a composition that has thefollowing constituents: β-1,3 (4)-endoglucanohydrolase, β-1,3 (4)glucan,diatomaceous earth, mineral clay and glucomannan. The composition isused in combination with a vaccine such that the ingestion of thecomposition by the animal enhances the effectiveness of the vaccine. Asdefined here, a vaccine stimulates the immune system, including theproduction of antibodies when administered to an animal. One indicationof an enhanced effectiveness of a vaccine is an increased titer ofantibodies to the vaccine antigen in the serum of the animal.

The combination can be used effectively in feed for individuals oranimals in many species such as mammals, including humans, and avians.In a preferred embodiment, the combinations are used in livestockmammals. The combinations can be used equally well with ruminants andnon-ruminants. Examples of ruminants include but are not limited tocattle, sheep, goats, cows, deer, bison and buffalo. Non-ruminantsinclude pigs, horses, sows and others. In a particularly preferredembodiment, the compositions of the composition are used for sheep andbovine livestock.

In one embodiment, the composition is fed to an animal during the periodwhen the animal is undergoing a vaccination protocol. A typicalvaccination protocol requires the administration of at least one andusually several doses of the vaccine over a defined period to maximizethe stimulation of the immune system and the production of antibodies.In a preferred embodiment, the composition is fed daily to an animalstarting before the initiation of the vaccination protocol andcontinuing after initiation of the vaccination protocol. In alternativeembodiments, the composition may be fed to an animal starting after theinitiation of the vaccination protocol or simultaneously with theinitiation of the protocol.

The vaccine of the combination may elicit a response to a pathogen, atoxin, a drug or other molecules. The vaccine may be a DNA vaccine wherea DNA molecule encoding an antigen is injected into an animal, resultingin synthesis of the antigen and subsequently an immune response to theantigen. In a preferred embodiment, the vaccine of the combinationstimulates the production of antibodies to a disease causing pathogen.In a particularly preferred embodiment, the vaccine stimulates theproduction of antibodies against pathogens that cause mastitis. The J5vaccine is one such commercially available mastitis vaccine (availablefrom Pfizer).

Other examples of pathogens and diseases for which humans and animalsare commonly vaccinated and which may benefit from a protocol whichenhances efficacy include, but are not limited to, infectious bovinerhinotracheitis (IBR), parainfluenza type 3 (PI3), bovine virus diarrheavirus (BVDV), bovine respiratory syncytial virus (BRSV), rota virus,corona virus, Campylobacter spp., Pasteurella spp., pinkeye, Salmonellaspp., Clostridium spp., Leptospirosis, Brucellosis, Newcastle disease,fowl pox, erysipelas, fowl cholera, Marek's Disease Virus (MDV),Infectious Bronchitis Virus (IBV), Avian encephalomyelitis, coccidiosis,rhinopneumonitis, equine influenza, Streptococcus equi, equine viralarteritis, equine monocytic ehrlichiosis, encephalomyelitis, West Nileencephalitis, rabies, parvovirus, adenovirus, Bordetella, Lyme disease,Giardia, pertussus, measles virus, hepatitis A and B, diphtheria, andpoliomyelitis.

The constituents of the composition of the combination are prepared bymethods commonly known in the art and can be obtained from commercialsources. The β-1,3 (4)-endoglucanohydrolase is produced from submergedfermentation of a strain of Trichoderma longibrachiatum. Thediatomaceous earth is available as a commercially-available acid-washed,product with 95% silica (SiO₂) and with its remaining components notassayed but consisting primarily of ash (minerals) as defined by theAssociation of Analytical Chemists (AOAC, 2002). β-1,3 (4)glucan andglucomannan can be from commercial preparations of yeast cell wallextract derived from primary inactivated yeast (Saccharomycescerevisiae) with the chemical composition shown in Table 1:

TABLE 1 Moisture 2-3% Dry matter 97-98% Proteins 14-17% Fat 20-22%Phosphorus 1-2% Mannans 22-24% β-1,3(4) glucans 24-26% Ash 3-5%The mineral clays (aluminosilicates) used in this composition may be anyof a variety of commercially-available clays including, but not limitedto, montmorillonite clay, bentonite and zeolite.

In a preferred embodiment of the combination, β-1,3(4)-endoglucanohydrolase, diatomaceous earth, glucan and glucomannan,and mineral clay are combined weight to weight in the ranges from about0.05-3%, 1-40%, 1-20% and 40-92%, respectively. In another preferredembodiment, 0-1,3 (4)-endoglucanohydrolase, diatomaceous earth, glucanand glucomannan, and mineral clay are combined at 0.1-3%, 5-40%, 2-15%and 40-80%, respectively. In an especially preferred embodiment, β-1,3(4)-endoglucanohydrolase, diatomaceous earth, glucan and glucomannan,and mineral clay are combined at 0.2-3%, 20-40%, 4-10% and 50-70%,respectively.

In one embodiment, the composition is a dry, free-flowing powder whichis suitable for direct inclusion into a commercially-available feed,food product or as a supplement to a total mixed ration or diet. Thepowder may be mixed with either solid or liquid feed or with water. Inanother embodiment, the composition is formed into pellets.

In one embodiment, when incorporated directly into feeds, thecomposition may be added in amounts ranging from about 0.1 to about 20kg per ton (2000 pounds) of feed. In a preferred embodiment, thecomposition is added to animal feedstuffs or to food in amounts fromabout 0.5 kg to about 10 kg per ton of feed. In an especially preferredembodiment, the composition may be added to feeds in amounts rangingfrom about 1 to about 5 kg per ton of feed.

When expressed as a percentage of dry matter of feed, the presentcomposition may be added to animal feedstuffs or to foods in amountsranging from about 0.01 to about 2.5% by weight, preferably from about0.0125% to about 2% by weight. In a preferred embodiment, thecomposition is added to animal feedstuffs or to food in amounts fromabout 0.05 to about 1.5% by weight, preferably from about 0.0625% toabout 1% by weight. In an especially preferred embodiment, the presentcomposition is added in amounts from about 0.1 to about 0.7% by weight,preferably from about 0.125% to about 0.5% by weight of feed.

Alternatively, the composition of the present combination may be feddirectly to mammalian or avian animals as a supplement in amounts offrom about 0.01 gram to about 1 gram per kilogram of live body weight,preferably from about 0.012 gram to about 0.5 gram per kilogram of livebody weight, more preferably from about 0.016 gram to about 0.37 gramper kilogram of live body weight per day. In an especially preferredembodiment, the composition may be provided for use with many species inamounts of from about 0.05 grams to about 0.20 grams per kilogram oflive body weight per day.

As examples, the composition may be provided to sheep in the range offrom about 2 grams per head per day to about 8 grams per head per day.For bovine animals, the composition may be provided in the range of fromabout 10 grams per head per day to about 60 grams per head per day. Oneof skill and art can appreciate that the amount of the composition fedcan vary depending upon the animal species, size of the animal and typeof the feedstuff to which the composition is added.

Examples are now provided in order to illustrate the concepts of thecomposition with a certain degree of specificity.

Example 1

An experiment was conducted with sheep with the goal of determining theability of the composition to increase expression of neutrophilL-selectin, a marker of the innate immune system, in immunosuppressedanimals. Animals (six per group) were divided into two groups: Controland Experimental. The Control group received a high energy rationconsisting of chopped hay available ad libitum, 1 lb of ground corn perhead per day and one lb of baked wheat mill run per head per day for aperiod of 28 days. During this time, they also received twice dailyinjections of dexamethasone, an immunosuppressive drug. The Experimentalgroup received daily intake of the composition (5 grams per head perday) for 28 days and received the same diet and dexamethasone injectionprotocol as the Control. This composition of the Experimental group was65.8 weight percent of mineral clay, 0.20 weight percent ofendoglucanohydrolase, 9.0 weight percent of glucans and glucomannan, and25 weight percent of calcined diatomaceous earth. At the end of thestudy, blood samples were recovered and neutrophils were purified usingPercoll gradient centrifugation. The amounts of L-selectin expression inneutrophils were assessed using Western blotting techniques andantibodies specific for L-selectin.

As shown in FIG. 1, top panel, animals that did not receive thecomposition had low and variable expression of L-selectin. As shown inFIG. 1, lower panel, animals that received the composition demonstrateda consistent increase in L-selectin expression. The top panel representssix Control, immunosuppressed animals. The lower panel represents sixExperimental immunosuppressed animals which received the composition intheir diet.

Example 2

In this study, the stimulation of the innate immune system in sheep wasexamined when the Experimental composition of Example 1 was provided ina pelleted diet. The basal diet consisted of 21.55% barley, 10.0% canolameal, 5% distillers grains, 40% ground corn, 1.50% limestone, 0.01%manganese sulfate, 0.01% microvitamin E, 4.0% molasses, 0.25% mono-cal,0.25% potassium chloride, 0.60% sodium chloride, 0.03% sodium selenite,15.79% wheat mill run, 0.01% zinc sulfate, 0.75% ammonium sulfate and0.25% cobalt sulfate. When the Experimental composition was added tothis diet, it was included at 0.6% replacing that portion of wheat millrun. Twenty-eight sheep were assigned to four treatments which consistedof a Control group, a group which received the Experimental compositionin powdered form, a group which received the Experimental composition inpelleted form where pellets were formed at a temperature of 160° F., anda group which received the Experimental composition in pelleted formwhere pellets were formed at 180° F. All animals were immunosuppressedvia daily injection of Dexamethasone.

The study was conducted using methods identical to Example 1 except thecomposition was administered in pellets that were manufactured byforming the pellets at high temperatures. The rationale for conductingthis study was to determine whether heating of the composition (as isrequired in pellet formation) might inactivate the ability of thecomposition to augment innate immunity. As shown in FIG. 2, sheep(Control) which did not receive the composition expressed very lowlevels of L-selectin in neutrophils. The provision of the Experimentalcomposition even in a pelleted (heated) form still increased expressionof neutrophil L-selectin markedly.

In FIG. 2, the uppermost panel represents neutrophil L-selectinexpression in immunosuppressed animals fed a control diet without thecomposition. The second panel (Powder) represents L-selectin expressionin immunosuppressed animals which received the Experimental compositionin unheated freely-mixed form as in Example 1 (Experimental group).Panels three and four represent neutrophil L-selectin expression inimmunosuppressed animals which received the Experimental composition inpelleted forms. The pellets used in Panel 3 were formed by heating to160° F. and Panel 4 pellets were heating to 180° F. during manufactureof the feeds.

Example 3

An experiment was performed with rats to investigate whether thecomposition had ability to augment innate immunity in a non-ruminantmodel. In this study, rats were assigned to one of two treatments: aControl group (un-supplemented diet) and an Experimental group where thecomposition of Example 1 was added to the diet at 1% of dry weight offeed. In this experiment, rats were fed a commercial ground rat chowwith or without the Experimental composition. Immunosuppression usingdexamethasone injection protocols were not utilized in this study.Following 14 days, blood samples were taken from anesthetized rats viacardiac puncture. Neutrophils were isolated from blood samples usingPercoll gradient centrifugation and total RNA was isolated using TriZol.

The concentration of the messenger RNA (mRNA) encoding rat L-selectin inthe neutrophil RNA samples was then determined by quantitative reversetranscriptase polymerase chain reaction (QRT-PCR) using primers whichwere specifically developed for assay of rat L-selectin. The amounts ofL-selectin mRNA were standardized by showing them as a proportion ofβ-actin mRNA, which is expressed in all cells at a fairly constantlevel. As shown in FIG. 3, and in agreement with the results in Examples1 and 2, the composition increased expression of L-selectin mRNA bygreater than 6-fold (P<0.05).

This study demonstrated that the increased expression of L-selectinprotein as shown in by Western blotting in Examples 1 and 2 may becaused by an increase in the mRNA encoding this protein. This impliesthat the composition alters the rate of transcription of the geneencoding L-selectin.

Example 4

Neutrophils, cells of the innate immune system, are able to signal andthereby up-regulate the production of antibodies by the acquired immunesystem through the secretion of interleukin-1β (IL-1β). To investigatethe ability of the composition to induce neutrophils to increasesynthesis of IL-1β, the concentration was assessed of IL-1β inneutrophils taken from the same sheep as described in Example 1. Tocomplete this study, Western blotting and antibodies specific for IL-1βwere used.

As shown in FIG. 4, animals which did not receive daily provision of thecomposition contained virtually undetectable levels of IL-1β; however,provision of the composition to animals caused a marked increase in theexpression of IL-1β (P<0.05). In FIG. 4, the top panel represents sixControl-fed immunosuppressed animals. The lower panel represents sixExperimental composition-fed immunosuppressed animals which received thecomposition. Concentrations of IL-1β were determined using Western blotanalysis and an antibody specific for IL-1β.

These data indicate that the composition possesses the ability to notonly increase markers of innate immunity (e.g., L-selectin; Examples 1,2 and 3) but to also increase expression of the key signaling molecule(i.e., IL-1β) that up-regulates the adaptive immune system.

Example 5

The goal of this experiment was to determine which genes weredifferentially-expressed in neutrophils following the feeding of thecomposition to peri-parturient dairy cattle. In this study, themechanism(s) by which the composition increased the expression of IL-1βin neutrophils was examined. Peri-parturient dairy cattle are a goodmodel because the stress of pregnancy leads to immunosuppression, makingthe cows particularly susceptible to infection.

In this experiment eight peri-parturient dairy cattle were assigned to aControl diet that did not have the Experimental composition and eightcattle were assigned to an Experimental group that received thecomposition of Example 1 in their diet (56 grams per day per head).Animals were fed the diets for approximately 28 days until parturition.At 12-15 hours following parturition, 500 ml samples of blood wererecovered via jugular puncture and neutrophils were prepared vialarge-scale Percoll gradient centrifugation.

RNA was isolated from neutrophils using the TriZol method and thenreverse-transcribed into cDNA using reverse transcriptase. Duringreverse transcription, differently-colored nucleotide-based dyes (Cy3and Cy5) were employed such that complementary DNAs (cDNAs) synthesizedfrom the two different treatment (Control and Experimental) groupsincorporated different colors. The cDNA samples from Experimental andControl groups were then applied to a BoTL-5 microarray slide. Thismicroarray was prepared at the Center for Animal Functional Genomics atMichigan State University and contains 1500 genes (each arrayed intriplicate) upon a glass slide. The cDNAs generated from theExperimental and Control group samples were then allowed to compete forbinding to the 1500 genes on the array and the relative expression ofthe genes was then assessed by comparing relative abundance of Cy3 andCy5 signals on each spot on the array. Data were then statisticallyanalyzed to identify those genes which were differentially-expressed(those genes where P<0.05).

The results showed that greater than 20 genes were differentiallyexpressed (P<0.05) in bovine neutrophils taken from the Experimentalgroup. Interleukin-converting enzyme (ICE) was one such up-regulatedgene. This was confirmed using QRT-PCR and primers specific to thebovine ICE sequence. ICE is the rate-limiting enzyme in the conversionof inactive pro-IL-1β to the active, secreted IL-1β. Thus, thecomposition may up-regulate adaptive immunity (i.e., such as increasingantibody titer) through its ability to increase expression of neutrophilICE activity and, consequently, secretion of IL-1β.

Example 6

To test the hypothesis that the composition enhanced the effectivenessof a vaccine, the development of titer following a vaccination protocolwas examined. Eighteen beef cattle (six cattle in each group) wereassigned to one of three treatments: Control, Treatment 1, (15 grams ofcomposition in the diet per head per day) and Treatment 2 (30 grams ofcomposition per head per day). The diet of the animals consisted of agrass hay diet which was offered ad libitum with a daily supplementwhich provided 14% crude protein, 3% crude fat and 20% crude fiber(Table 2). Animals were provided with 12 pounds of this supplement perhead per day throughout the trial. The Experimental composition ofExample 1 was mixed directly into this supplement so that expectedintakes of 15 grams per head per day and 30 grams per head per day weredelivered to cattle in Treatment 1 and 2 respectively.

The animals were administered these diet treatments for 56 days afterwhich the composition was withdrawn from the diet of Treatments 1 and 2.All animals were maintained on the same control diet without thecomposition until Day 84. On days 7, 21 and 35 of the experiment allanimals were administered an E. coli J5 vaccine (Pfizer). Thisvaccination protocol (i.e., three injections, 14 days apart) followedmanufacturer's recommendations. This vaccine is used commercially in thedairy industry as a means of reducing the likelihood of coliformmastitis. A limitation to this vaccine, and to most other vaccines, isthe variable and limited response in titer.

Blood samples were taken using jugular puncture on Days 0, 14, 28, 42and 56 on which day the composition was removed from the animals' diet.All animals remained on a common diet without the composition until day84. Blood samples were also taken on Day 82 to determine whether anychanges in titer induced by the composition were maintained followingits withdrawal from the diet. Serum was prepared from all animals bycentrifugation. Concentrations of antibodies specific for the E. coli J5vaccination were assessed in three different immunoglobulin fractions(IgM, IgG1 and IgG2) using enzyme-linked immunosorbant assays (ELISAs).To assay E coli J5 titers in the IgM, IgG1 and IgG2 immunoglobulinfractions, a culture of E. coli (obtained from Dr. Jeanne Burton, Centerfor Animal Functional Genomics, Department of Animal Sciences, MichiganState University) was grown, harvested and used to coat 96-well plates.Subsequently, serum samples (diluted 1:5000) from the animals were addedto individual wells on the coated plates and allowed to incubate for onehour to allow the antibodies in the animal serum to bind to the E. coliJ5 antigen. The ELISA plates were washed with phosphate-buffered saline(PBS) containing Tween-20.

Secondary antibodies (horseradish peroxidase (HRP)-conjugated ovineanti-bovine) that bound specifically either to bovine IgM, IgG1 or IgG2(Beth Laboratories, Montgomery, Tex.) were added to the washed plates.The secondary antibodies were conjugated to horseradish peroxidase.Following incubation for an additional one hour with the secondaryantibodies, the plates were washed again with PBS and Tween-20. Theperoxidase substrate TMB (tetramethylbenzidine) was added to the platesand the resulting color reaction was quantitated by measuring theabsorbance at 450 nm using an ELISA plate reader.

As shown in Table 3, the Experimental composition had no effect (at thethreshold of P>0.05) on the development of E. coli J5 titer associatedwith the IgM antibody isotype. However, the composition stimulated andmaintained J5 titer in IgG1 and IgG2 antibody isotypes as shown in FIGS.5 and 6 and Tables 4 and 5.

The IgG1 titer increased on Day 56 (P<0.05) in animals which hadreceived both Treatment 1 and Treatment 2 (Table 4 and FIG. 5). By Day82, titer in Control animals had fallen back to pre-experiment (Day 0)levels. However, the animals which had been fed Treatment 1 or Treatment2 had elevated J5 titer compared to the Control animals (P<0.05). Infact, animals which had received the Treatment 1 or 2 demonstrated noloss of IgG1 titer once the invention had been removed from the dietfollowing 56 days (Table 4 and FIG. 5).

As shown in Table 5 and FIG. 6, with respect to the IgG2 fraction,addition of 15 g per head per day (Treatment 1) or 30 g per head per day(Treatment 2) of the composition caused a step-wise increase in J5 IgG2titer at 42 days (i.e. the higher dose increased titer by 57% and thelow dose by 30% compared to the Control), although this effect was notstatistically significant at P<0.05 (the P value was 0.16). Following 56days, Treatment 2, (the higher dose of the Composition) caused asignificant elevation in J5 titer (P<0.05). Treatment 1 (the low dose ofthe Composition) caused an elevation in J5 titer within the IgG2fraction at this time point although this effect was not statisticallysignificant at a threshold of P<0.05 (P value=0.12). On Day 82 of thestudy, after the Composition had been withdrawn from the diet, animalsfed the higher dose of the Composition had an elevated titer within theIgG2 fraction (14% increase compared to the Control); however, thiseffect was not significant at the threshold of P<0.05 (P value is=0.09).

These data indicate that the Composition has the ability to increasetiter of and, furthermore, to maintain titer in the IgG1 fractionfollowing withdrawal of the composition from the diet (P<0.05). TheComposition also has the ability to increase development of titer withinthe IgG2 fraction (P<0.05).

IL-1β concentrations were also examined in the serum samples from theControl, Treatment 1 and Treatment 2 animals using an ELISA for IL-1β(R+D Systems Minneapolis, Minn.)). As shown in FIG. 7, results indicatedthat both Treatment 1 and Treatment 2 increased serum concentrations ofIL-1β after 28 days. After 56 days, the low dose of the Composition alsosignificantly elevated serum concentration of IL-1β (P<0.05). The highdose of the Composition caused a numerical elevation in serum IL-1β onDay 56 of the study; however, this effect was not significant at P<0.05(P value=0.23). Although not wishing to be bound by theory, thecomposition may stimulate the innate immune system (e.g., via neutrophilactivation) and neutrophils then stimulate the acquired immune system bysecretion of IL-1β. IL-1β specifically increased ability of B-cells toincrease the rate of development of IgG2 titer and maintains titerwithin the IgG1 fraction.

Protein concentrations of neutrophil L-selectin were also assessed usingWestern blotting as shown in FIG. 8. The mRNA concentration forL-selectin was also determined as for the rat study (Example 4) butusing bovine-specific primers. It was determined that both the Treatment1 (15 g of Experimental composition/head/day) and Treatment 2 (30 g ofExperimental composition/head/day) increased (P<0.05) concentrations ofneutrophil L-selectin compared to the Control treatment.

TABLE 2 Ingredient percent of supplement (as feed basis) Cotton hulls32.5 Corn 25.0 Wheat mill run 6.25 Canola meal 7.50 Distillers grains8.73 Limestone 0.75 Dynamate 0.25 Magnesium oxide 0.15 Alfalfa meal 14.0Urea 0.50 Rumensin 0.01 Zinc sulphate 0.01 Selenium 0.04 Sodium chloride0.30 Molasses 4.00

TABLE 3 Control Treatment 1 Treatment 2 Day of study (Abs at 450 nm)(Abs at 450 nm) (Abs at 450 nm) 14 days 0.477 0.404 0.391 28 days 0.7360.409 0.424 42 days 0.842 0.405 0.408 56 days 1.97 0.717 1.65

TABLE 4 Control Treatment 1 Treatment 2 Day of study (Abs at 450 nm)(Abs at 450 nm) (Abs at 450 nm)  0 days 0.035 0.036 0.033 28 days 0.0650.045 0.035 42 days 0.058 0.065 0.109 56 days 0.238^(a) 0.543^(b)0.471^(b) 82 days 0.033^(a) 0.549^(b) 0.573^(b) Values within a rowwhich do not share a common superscript differ significantly (P < 0.05threshold).

TABLE 5 Control Treatment 1 Treatment 2 Day of study (Abs at 450 nm)(Abs at 450 nm) (Abs at 450 nm)  0 days 0.079 0.104 0.091 28 days 0.2200.229 0.215 42 days 0.228 0.296 0.358 56 days 0.306^(a) 0.401^(a)0.681^(b) 82 days 0.178 0.179 0.252 Values within a row which do notshare a common superscript differ significantly (P < 0.05 threshold).

Example 7

An experiment was completed with sheep to investigate the ability of theExperimental composition of Example 1 to augment adaptive immunity inresponse to the J5 E. coli vaccination protocol which was employed inExample 6. Animals (twelve animals per treatment) were allotted to threetreatments: Control, Treatment 1 (3 grams per head per day of theExperimental composition of Example 1) or Treatment 2 (6 grams per headper day of the Experimental composition of Example 1). Animals were fedthese diets for 75 days and were vaccinated with the Pfizer J5 vaccineon Days 7, 21 and 35 of the trial. Blood samples were taken on Days 0,35 and 75. Blood samples were also taken on Day 90 after the Treatment 1or Treatment 2 composition had been withdrawn from the diet to determinewhether this composition had the ability to maintain titer.

In this study, a secondary antibody (HRP-conjugated rabbit anti-ovineIgG: Beth Laboratories, Montgomery, Tex.) which recognized both IgG1 andIgG2 sheep isotypes was used. Table 6 and FIG. 9 show data from thecombined sheep IgG1 and IgG2 fractions. The methods used in this studyto conduct the ELISA were identical to those outlined in Example 6except that an anti-ovine IgG antibody (VMRD, Pullman, Wash.) was usedinstead.

On Day 35, the high dose of the Experimental composition caused anelevation in the total of the IgG1 and IgG2 titers. On Day 75, the lowand high doses of the Composition caused a step-wise increase in J5titer. Specifically, the low dose caused a 17% increase in titer and thehigh dose caused a 31% increase in J5 titer. On Day 90, animals inTreatment 1 did not have an elevation in titer compared to control-fedanimals. However, animals in Treatment 2 which received the high dose ofthe Composition exhibited a 24% elevation in J5 titer.

These data support the observations of Experiment 6 in thatadministration of the Experimental composition increased development oftiter within the IgG1 and IgG2 fraction and maintained titer followingwithdrawal of the Experimental composition from the diet for anadditional 15 days.

TABLE 6 Day Control (Abs U at Treatment 1 (Abs U Treatment 2 (Abs U ofstudy 450 nm) at 450 nm) at 450 nm)  0 days 0.174 0.173 0.173 35 days0.270 0.285 0.336 75 days 0.350 0.410 0.460 90 days 0.376 0.379 0.469The invention tended to increase J5 titer in the combined IgG fractionsalthough the P values were greater than the P < 0.05 threshold.

It will be understood that the embodiments of the present compositionwhich have been described are illustrative of some of the applicationsof the principles of the present composition. Numerous modifications maybe made by those skilled in the art without departing from the truespirit and scope of the composition. Various features which aredescribed herein can be used in any combination and are not limited toprocure combinations that are specifically outlined herein.

We claim:
 1. A combination for enhancing vaccine effectiveness,comprising: a vaccine; and a composition comprising β-glucans, β-1,3(4)-endoglucanohydrolase, silica, a mineral clay, and mannans, whereinthe composition increases a titer of antibodies to the vaccine relativeto the titer of the antibodies in the absence of the composition.
 2. Thecombination of claim 1 wherein the composition is added to feed ofanimals in the range of from about 0.01% to about 2.5% by weight of thefeed on a dry weight basis.
 3. The combination of claim 1 wherein thecomposition is added to feed of animals in the range of from about 0.01gram per kilogram of body weight of the animal to about 1 gram perkilogram of body live weight of the animal.
 4. The combination of claim1 wherein the composition is administered to bovine livestock in therange of from about 10 grams to about 60 grams per day per animal. 5.The combination of claim 1 wherein the composition is administered tosheep in the range of from about 2 grams to about 10 grams per day peranimal.
 6. The combination of claim 1 wherein the vaccine stimulates theproduction of antibodies to one or more pathogens that causes mastitis.7. The combination of claim 1 wherein the vaccine is a mastitis vaccine.8. The combination of claim 1 wherein the composition increases a titerof IgG1 or IgG2 antibodies relative to the titer of the IgG1 or IgG2antibodies in the absence of the composition.
 9. The combination ofclaim 8 wherein the increased titer of the IgG1 or IgG2 antibodiesrelative to the titer of the IgG1 or IgG2 antibodies in the absence ofthe composition is maintained after the withdrawal of the compositionfrom the diet of the animals.
 10. The combination of claim 1 wherein thesilica is obtained from diatomaceous earth.
 11. The combination of claim1 wherein the mannans comprise glucomannan.
 12. A method for vaccinatingan individual, comprising: administering the combination of claim 1 toan individual of a mammalian species or an avian species, whereinadministering the combination comprises administering the composition tothe individual prior to administering a dose of the vaccine to theindividual, and vaccinating the individual with the vaccine, wherein theindividual subsequently has an increased titer of antibodies to thevaccine relative to a titer of antibodies to the vaccine if thecomposition had not been administered.
 13. The method of claim 12wherein the composition is administered daily to the individual.
 14. Themethod of claim 12 wherein the composition is added to feed of theindividual in the range of from about 0.01% to about 2.5% by weight ofthe feed on a dry weight basis.
 15. The method of claim 12 wherein thecomposition is added to feed of the individual in the range of fromabout 0.01 gram per kilogram of live body weight of the individual to 1gram per kilogram of live body weight.
 16. The method of claim 12wherein the individual subsequently has an increased titer of IgG1 orIgG2 antibodies relative to a titer of the IgG1 or IgG2 antibodies ifthe composition had not been administered.
 17. The method of claim 12wherein the composition is administered daily for at least 7 days priorto administering the dose of the vaccine.
 18. The method of claim 12,further comprising administering the composition after administering thedose of the vaccine and prior to administering one or more subsequentdoses of the vaccine.
 19. The method of claim 12, wherein thecomposition is mixed with solid feed, liquid feed, or water.
 20. Themethod of claim 12 wherein the vaccine is a vaccine for mastitis,infectious bovine rhinotracheitis, parainfluenza type 3, bovine virusdiarrhea virus, bovine respiratory syncytial virus, rota virus, coronavirus, Campylobacter spp., Pasteurella spp., pinkeye, Salmonella spp.,Clostridium spp., Leptospirosis, brucellosis, Newcastle disease, fowlpox, erysipelas, fowl cholera, Marek's Disease Virus, InfectiousBronchitis Virus, avian encephalomyelitis, coccidiosis,rhinopneumonitis, equine influenza, Streptococcus equi, equine viralarteritis, equine monocytis ehrlichiosis, encephalomyelitis, West Nileencephalitis, rabies, parvovirus, adenovirus, Bordetella, Lyme disease,Giardia, pertussus, measles virus, hepatitis A, hepatitis B, diphtheria,or poliomyelitis.